Role of MCM10 in DNA Replication Initiation In Vitro

DNA复制是一个高度调控的过程。在细胞分裂过程中，每个细胞必须以极高的保真度快速复制其基因组。在真核生物中，染色体DNA复制的起始是通过在复制起始处的大量蛋白质的逐步组装而发生的。最早的步骤发生在细胞周期的G1期，是6亚基起始点识别复合体（ORC）与复制起始点的结合，随后是Cdc6、Cdt1和MCM2-7复合体。总的来说，这些蛋白质来自复制前复合体（pre-RC）。在G1期到S期的过渡过程中，Cdc7和Cdk2激酶激活pre-RC。在G1期到s期的过渡过程中，Cdc7和Cdk2激酶激活pre-RC，刺激Cdc45的募集，将pre-RC转化为一个活跃的复制叉。在酿酒酵母中的实验表明，在G1期参与预RC组装的一种蛋白质是MCM10。Walter实验室最近克隆了非洲爪蟾MCM10的同源物，并使用可溶性非洲爪蟾提取物表征了其功能，该提取物重建了真核生物DNA复制的步骤。出乎意料的是，MCM不需要预rc组装，但对于Cdc45加载步骤至关重要。本研究旨在阐明MCM10刺激CDC45加载的分子机制。此外，我将探讨MCM10是否可能是细胞周期检查点的目标。

DNA replication is a highly regulated process. During cell division each cell must replicate its genome rapidly, yet with extremely high fidelity. In eukaryotes, the initiation of chromosomal DNA replication occurs through the stepwise assembly of numerous proteins at origins of replication. The earliest step, which occurs in the G1 phase of the cell cycle, is the binding of the six-subunit origin, recognition complex (ORC) to replication origins, followed by Cdc6, Cdt1 and the MCM2-7 complex. Collectively, these proteins from the pre-replicative complex (pre-RC). During the transition from G1 to S phase, Cdc7 and Cdk2 kinases activate the pre-RC. During the transition from G1 to S-phase, Cdc7 and Cdk2 kinases activate the pre-RC stimulating the recruitment of Cdc45, converting the pre-RC into an active replication fork. Experiments in S. cerevisiae suggests that one protein involved in pre- RC assembly during G1 phase is MCM10. The Walter laboratory has recently cloned the Xenopus homolog of MCM10, and has characterized its function using a soluble Xenopus extract that reconstitutes the steps involved in eukaryotic DNA replication. Unexpectedly, MCM is not required for pre-RC assembly, but is essential for the Cdc45 loading step. The goal of this proposal is to elucidate the molecular mechanism by which MCM10 stimulates CDC45 loading. In addition, I will explore whether MCM10 may be the target of cell cycle checkpoints.